Cancer treatment composition and method using signal transduction modulators and natural plant essential oils as synergists for conventional treatments

ABSTRACT

Pharmaceutical compositions containing plant essential oil compounds, natural or synthetic, or mixtures or derivatives thereof, with one or more signal transduction modulators, for the prevention and treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional PatentApplication No 60/111,271, filed Dec. 7,1998, the entire disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates, in general, to therapeuticallyeffective pharmaceutical compositions containing conventional cancertreatments in combination with signal transduction modulators and/ornatural plant essential oils, and methods for using same forprophylactically or therapeutically treating of soft tissue cancer inmammals, including humans, such as, for example, breast cancer.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is a proliferative disease of mammary epithelialcells and estrogen has been shown to stimulate cell proliferation ofthese cells both in culture and in mice (Soto and Sonnenschein, 1985;Osborne, 1988). Xenoestrogens have been proposed to stimulate cellproliferation through binding and activating estrogen receptors (ERs)(Miller et al., 1993; Hoffman, 1992). The incidence of breast cancer hasbeen steadily rising during the past two or three decades, a trendcharacterized by increasing rates among estrogen-responsive tumors, bycontinuing increases among older women, and by growing numbers in bothdeveloped and developing countries (Harris et al., 1992). Between1973-1980, the incidence of breast cancer in the United States increaseda modest 8% among women under 50 years of age, while it rose 32.1% amongwomen in the age group of 50 years or older (Ries et al., 1991). Thisupward shift is consistent with the historical pattern of accumulationof organochlorine insecticides residues (xenoestrogens) in theenvironment (Mussalo-Rauhamaa et al., 1990; Wolff et al., 1993; Davis etal., 1993). Breast cancer is also the second leading cause of cancerdeaths in women and it is estimated that in 1998, there will be anadditional 43,900 deaths due to breast cancer. Environmental estrogensor endocrine disruptors have been suggested to play a role in theetiology or promotion of breast cancer (Davis et al., 1993; Dewailly etal., 1995). Experimental evidence reveals that xenoestrogens affectestrogen production and metabolism and are among the risk factors thatcause breast cancer (Nelson, 1974; Berthois et al., 1986; Henderson etal., 1993; Jobling et al., 1996; Dees et al., 1997). Most of the knownrisk factors for breast cancer, which at least account for 30% of cases(Henderson et al., 1993) are linked with total life-time exposure toreproductive chemicals such as estrogen and xenoestrogens.

[0004] It appears evident that soft tissue cancer in mammals isincreasing every year as a result of increased estrogen levels andincreased exposure to environmental xenoestrogens. For example, thenumber of prescriptions of estrogen for women in menopause is rapidlyincreasing, presently estimated at 50,000,000 prescriptions annually inthe United States alone. This increasing use of estrogen partiallyaccounts for the higher risk of breast cancer in both young andmiddle-aged women. Estrogen is present in all mammals and is essentialin women for reproductive organs such as ovary, uterus, breast, etc. Inmen, however, estrogen is required for sperm production and maturation.The abusive use of estrogen prescribed for women is at least partiallyresponsible for the development of soft tissue cancers, especiallybreast cancer. It is therefore desirable to antagonize or counteract theadverse effects of estrogen in women.

[0005] The current FDA-approved treatments, e.g., tamoxifen, in theUnited States are effective to some extent in some of the femalepopulation in antagonizing the adverse effects of estrogen.Unfortunately, these treatments are not totally effective and maythemselves cause additional health related effects, such as uterinecancer. Thus, if one could identify compounds that would make thecurrent treatments more effective, or would work in conjunction with, orin lieu of, the present treatments, it is possible some of these adverseside effects would be alleviated or even eliminated. Possible sources ofalternative treatments are natural, non-toxic compounds. It is proposedthat these compounds would advantageously provide for safer and moreeffective treatments.

[0006] The current literature suggests the use of certain signaltransduction modulators as treatments for breast and colon cancer. Theuse of certain monoterpenoid plant essential oils (alpha-terpineol,linalool, and limonene) is also suggested as a potential treatment forbreast cancer. These monoterpenoids however are not totally effectiveand have been proven to be weak anti-proliferative cancer products. Inaddition, these data do not suggest the capability of these compounds toantagonize the action of estrogen. This may raise the question of howthis product may interact in women with estrogen supplement. None of thedata disclose the synergistic effects of plant essential oils and/orsignal transduction modulators in combination with conventionaltreatments to enhance the anti-proliferative and anti-estrogenic effectsof the current treatments without the corresponding increase in relatedhealth risks.

[0007] Accordingly, there is a great need for novel compositionscontaining current anti-cancer treatments in combination with novelingredients to improve the effective treatment of soft tissue cancer andto reduce the adverse effects of such current anti-cancer treatments.

SUMMARY OF THE INVENTION

[0008] An object of the present invention is to provide novelcompositions containing beneficial mixtures of current treatments forsoft tissue cancer with non-toxic plant essential oils, or derivativesthereof, and/or one or more signal transduction modulators, for the safeand effective treatment of soft tissue cancer.

[0009] The above and other objects are accomplished by the presentinvention which is directed to novel pharmaceutical compositions forprophylactically or therapeutically treating soft tissue cancers. Theinventive compositions comprise at least one plant essential oilcompound and/or at least one signal transduction modulator in admixturewith a conventional cancer treatment, e.g., tamoxifen.

[0010] Additional objects and attendant advantages of the presentinvention will be set forth, in part, in the description that follows,or may be learned from practicing or using the present invention. Theobjects and advantages may be realized and attained by means of thefeatures and combinations particularly recited in the appended claims.It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not to be viewed as being restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The features and advantages of the present invention will becomemore clearly appreciated from the following description taken inconjunction with the accompanying drawings in which like elements aredenoted by like reference numerals.

[0012]FIG. 1 shows the concentration-response of tamoxifen and forskolinagainst estrogen-induced (E₂-induced) proliferation in breast cancercells.

[0013]FIG. 2 shows the mixed exposure effect of tamoxifen (TX) andgeldanamycin (GM) on E₂-induced growth in breast cancer cells.

[0014]FIG. 3 shows the mixed exposure effect of forskolin (FK) withtamoxifen (TX), and geldanamycin (GM) with TX on E₂-induced growth inbreast cancer cells.

[0015]FIG. 4 shows the time-course effect of tamoxifen (TX) alone and TXplus forskolin (FK) on E₂-induced growth in breast cancer cells. Datawas calculated as percentage of control value.

[0016]FIG. 5 shows the time-course effect of tamoxifen (TX) and TX plusforskolin (FK) on E₂-induced growth in breast cancer cells. Data iscalculated as percentage of E₂-induced cell growth.

[0017]FIG. 6 shows the mixed exposure effect of tamoxifen (TX, 1 μM) andforskolin (FK, 5 μM) on the growth of breast cancer cells.

[0018]FIG. 7 shows the mixed exposure effect of tamoxifen (TX, 1 μM) andforskolin (FK, 1 μM) on the growth of breast cancer cells.

[0019]FIG. 8 shows the effect of different concentrations ofgeldanamycin, the c-Src kinase inhibitor and tamoxifen (TX) on thegrowth of breast cancer cells.

[0020]FIG. 9 shows the mixed exposure effect of tamoxifen (TX, 5 μM) andmitogen activated protein kinase, MAPK, inhibitor, PD98059 (2 μM) on thegrowth of breast cancer cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0021] All patents, patent applications and literatures cited in thisdescription are incorporated herein by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, will prevail.

[0022] In a preferred embodiment, the present invention provides a novelpharmaceutical composition comprising at least one plant essential oilcompound derived from either natural or synthetic sources and at leastone signal transduction modulator including mixtures or derivatives ofplant essential oil compound. In a further preferred embodiment, thepresent invention is directed to a novel pharmaceutical compositioncomprising an anti-cancer treatment, such as tamoxifen, at least oneplant essential oil compound derived from either natural or syntheticsources or mixtures or derivatives plant essential oil compounds, and/orat least one signal transduction modulator.

[0023] The specific plant essential oils disclosed herein or derivativethereof comprise a monocyclic, carbocyclic ring structure havingsix-members and substituted by at least one oxygenated or hydroxylfunctional moiety. Examples of plant essential oils encompassed withinthe present invention, include, but are not limited to, members selectedfrom the group consisting of aldehyde C16 (pure), amyl cinnamicaldehyde, amyl salicylate, anisic aldehyde, benzyl alcohol, benzylacetate, cinnamaldehyde, cinnamic alcohol, a-terpineol, carvacrol,carveol, citral, citronellal, citronellol, p-cymene, diethyl phthalate,dimethyl salicylate, dipropylene glycol, eucalyptol (cineole), eugenol,iso-eugenol, galaxolide, geraniol, guaiacol, ionone, d-limonene,menthol, methyl anthranilate, methyl ionone, methyl salicylate,α-phellandrene, pennyroyal oil perillaldehyde, 1- or 2-phenyl ethylalcohol, 1- or 2-phenyl ethyl propionate, piperonal, piperonyl acetate,piperonyl alcohol, D-pulegone, terpinen4-ol, terpinyl acetate, 4-tertbutylcyclohexyl acetate, thyme oil, thymol, metabolites oftrans-anethole, vanillin, ethyl vanillin, and the like. As these plantessential oil compounds are known and used for a variety of otherpurposes, they may be prepared by a skilled artisan by employing knownmethods.

[0024] The above plant essential oil compounds can also be administeredin combination with at least one signal transduction modulator.Non-limiting examples of signal transduction modulators include membersselected from the group consisting of cyclic adenosine monophosphate(cAMP)/cAMP-dependent protein kinase, tyrosine kinase, calciumphospholipid-dependent protein kinase (PKC), mitogen activated proteinkinase family members, calcium-calmodulin-dependent protein kinase, andgrowth factor receptor inhibitors. Specific non-limiting examples ofsuch signal transduction modulators include octopamine, forskolin,PD98059, geldanamycin and genistein, and staurosporin. When the plantessential oil compounds of the present invention are administered incombination therapies with other agents, they may be administeredsequentially or concurrently to a patient in need of treatment.

[0025] The above plant essential oil compounds and signal transductionmodulators of the present invention may be purchased from conventionalsources, may be readily isolated from specific plants or trees andpurified (isolated) or may be synthesized using conventional techniques.Advantageously, these compounds may be conveniently synthesized fromreadily available starting materials. The relative ease with which thecompounds of this invention can be obtained represents an enormousadvantage in the large-scale production of these compounds.

[0026] It will be appreciated that the therapeutically-active plantessential oil compounds of the present invention may be modified orderivatized by appending appropriate functionalities, i.e., functionalgroups, to enhance selective biological properties. Such modificationsare known in the art and include those that increase biologicalpenetration into a given biological compartment (e.g., blood, lymphaticsystem, central nervous system), increase oral availability, increasesolubility to allow administration by injection, alter metabolism andalter rate of excretion. In addition, the plant essential oil compoundsmay be altered to pro-drug form such that the desiredtherapeutically-active form of the compound is created in the body ofthe patient as the result of the action of metabolic or otherbiochemical processes on the pro-drug. Some examples of pro-drug formsinclude ketal, acetal, oxime, and hydrazone forms of compounds whichcontain ketone or aldehyde groups.

[0027] Moreover, the therapeutically-effective plant essential oilcompounds of the present invention may contain one or more asymmetriccarbon atoms and thus may occur as racemates and racemic mixtures,single enantiomers, diastereomeric mixtures and individualdiastereomers. Each stereogenic carbon may be of the R or Sconfiguration. All such isomeric forms of these compounds are expresslyincluded within the purview of the present invention.

[0028] As will be appreciated, the compositions and method of thepresent invention include pharmaceutical compositions that comprise atleast one plant essential oil, and pharmaceutically acceptable saltsthereof, in combination with any pharmaceutically acceptable carrier,adjuvant or vehicle. The term “pharmaceutically acceptable carrier oradjuvant” refers to a carrier or adjuvant that may be administered to apatient, together with a plant essential oil compound of the presentinvention, and which does not destroy the pharmacological activitythereof and is nontoxic when administered in doses sufficient to delivera therapeutic amount of the compound.

[0029] Pharmaceutically acceptable salts of the plant essential oilcompounds of this invention include those derived from pharmaceuticallyacceptable inorganic and organic acids and bases. Examples of suitableacid salts include, without limitation, acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate,glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate andundecanoate. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

[0030] Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(C₁₋₄alkyl)4+salts. The present invention also envisions the quatemization ofany basic nitrogen-containing groups of the compounds disclosed herein.Water or oil-soluble or dispersible products may be obtained by suchquaternization.

[0031] Further, pharmaceutically acceptable carriers, adjuvants andvehicles that may be used in the pharmaceutical compositions of thisinvention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug delivery systems(SEDDS) such as d.alpha-tocopherol polyethyleneglycol 1000 succinate, orother similar polymeric delivery matrices or systems, serum proteins,such as human serum albumin, buffer substances such as phosphates,glycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes, such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances,polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycoland wool fat. Cyclodextrins such as alpha-, beta-, andgamma-cyclodextrin, or chemically modified derivatives such ashydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-.beta.-cyclodextrins, or other solublized derivativesmay also be advantageously used to enhance delivery oftherapeutically-effective plant essential oil compounds of the presentinvention.

[0032] The pharmaceutical compositions of this invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir,however, oral administration or administration by injection ispreferred. The-pharmaceutical compositions of this invention may containany conventional non-toxic pharmaceutically-acceptable carriers,adjuvants or vehicles. In some cases, the pH of the formulation may beadjusted with pharmaceutically acceptable acids, bases or buffers toenhance the stability of the formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

[0033] The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as Ph. Helv or a similar alcohol.

[0034] The pharmaceutical compositions of the present invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, and aqueous suspensions andsolutions. In the case of tablets for oral use, carriers which arecommonly used include lactose and corn starch. Lubricating agents, suchas magnesium stearate, are also typically added. For oral administrationin a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening and/or flavoring and/or coloring agents maybe added.

[0035] The pharmaceutical compositions of the present invention may alsobe administered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycol.

[0036] Although rare, topical administration of the pharmaceuticalcompositions of the present invention is especially useful when thedesired treatment involves areas or organs readily accessible by topicalapplication. For application topically to the skin, the pharmaceuticalcomposition should be formulated with a suitable ointment containing theactive components suspended or dissolved in a carrier. Carriers fortopical administration of the compounds of this invention include, butare not limited to, mineral oil, liquid petroleum, white petroleum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, the pharmaceutical composition can beformulated with a suitable lotion or cream containing the activecompound suspended or dissolved in a carrier. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyidodecanol, benzyl alcoholand water. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topically-transdermalpatches are also included in this invention.

[0037] The pharmaceutical compositions of this invention may beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

[0038] Another acceptable pharmaceutical preparation would be anencapsulated form of the plant essential oil compounds, as is, ormodified as per the prior descriptions. The walls of the capsules couldbe designed to release the plant essential oils rapidly, i.e. oneminute, hour or day, or it could be designed to release over somedesignated period of time, i.e. days, weeks or months. The wallmaterials could be natural or synthetic is polymers acceptable to the USFDA or composed of lipids or other suitable materials.

[0039] These capsules could be delivered either orally or by injectionand could be either water or oil based depending upon the desired methodof use or required rate of release.

[0040] Dosage levels of between about 0.001 and about 100 mg/kg bodyweight per day, preferably between about 0.5 and about 75 mg/kg bodyweight per day of the active ingredient compound are useful in theprevention and treatment of soft tissue cancers. Typically, thepharmaceutical compositions of this invention will be administered fromabout 1 to about 5 times per day or alternatively, as a continuousinfusion. Such administration can be used as a chronic or acute therapy.The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 5% to about 95% active compound(w/w). Preferably, such preparations contain from about 20% to about 80%active compound.

[0041] Upon improvement of a patient's condition, a maintenance dose ofa compound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

[0042] The prophylactic use of the present invention may require thedaily intake of a prophylactically-effective amount.

[0043] As the skilled artisan will appreciate, lower or higher dosesthan those recited above may be required. Specific dosage and treatmentregimens for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health status, sex, diet, time ofadministration, rate of excretion, drug combination, the severity andcourse of a cancer, the patient's disposition to cancer and the judgmentof the treating physician.

[0044] The compositions and methods of the present invention will befurther illustrated in the following non-limiting Examples that show thesynergistic effects of signal transduction modulators with tamoxifen.When reading the following examples, it will be appreciated that thegrowth and proliferation of MCF-7 cells are strictly estrogen-dependent.In the presence of estrogen, the cells grow, confluent and form foci,the landmark of tumor diagnosis. In the absence of estrogen, the growthof these cells is slow and the formation of foci is rare. The followingExamples are illustrative of various embodiments only and do- not limitthe claimed invention regarding the materials, conditions, weightratios, process parameters and the like recited herein. Examples 14exemplify, inter alia, the present invention against breast cancer inmenopausal women who are taking estrogen supplement. Examples 5-7exemplify, inter alia, the present invention against breast cancer inpre-menopausal women and/or elderly women who are not taking estrogensupplement, but have potential risk factors to develop breast cancer.

Example 1

[0045] Dose-Response of Tamoxifen on Estrogen (E₂)-Induced CellProliferation MCF-7 cells were cultured in growth DMEM mediumsupplemented with 10% fetal bovine serum (FBS). At 85% confluence, cellswere sub-cultured in 5% FBS serum stripped medium, phenol red free for24 hours prior to the treatment of the test chemical. Cells were treatedwith 10 nM E₂ (=2.7 ng E₂/ml) with and without different concentrationsof tamoxifen (TX) 24 hours prior to cell proliferation assessment. Cellproliferation was measured using ³H-thymidine incorporation. The studywas done in triplicate and a control was used with solvent only. Controlreceived solvent only at <0.1% ethanol.

[0046] Results are shown in Table 1. In Table 1, percentage of controlis calculated either based on the control (solvent only, ethanol 0.1%)or based on the E₂ values which is between parenthesis. TABLE 1Dose-Response Effect of TX ³H-thymidine incorporation Test dpm/μg % of %Treatment Conc. dpm protein control protection Control  1993 306.6 100(solvent only) E₂ 10 nM 16388 483.4 157 proliferation TX  1 μM  2586129.3  42 58 TX + E₂ 12336 379.6 124 (79) proliferation (21) TX  5 μM 1848 112.0  36 64 TX + E₂  4007 260.2  85 (53) 15 (47) TX 10 μM  1545 99.8  33 67 TX + E₂  3239 118.5  39 (25) 61 (75)

[0047] Table 1 demonstrates that the antagonistic action of TX againstthe E₂-induced cell proliferation is dose-dependent. TX at lowconcentrations between 0.56 ug/ml to 2.8 ug/ml (=1 - 5 μM) is potentagainst the E₂-induced cell proliferation.

Example 2

[0048] Dose-response of Signal Transduction Modulators on E2-InducedCell Proliferation To determine the impact of two key signalingproteins, cAMP/PKA system and tyrosine kinase family member, c-Srckinase (p^(src60)) on estrogen-induced cell proliferation in humanepithelial breast cancer cells, MCF-7 cells were cultured in growth DMEMmedium supplemented with 10% fetal bovine serum (FBS). At 85%confluence, cells were sub-cultured in 5% FBS serum stripped medium,phenol red free for 24 hours prior to the treatment of the testchemical. Cells were treated with 10 nM E₂ (=2.7 ng E₂/ml) with andwithout different concentrations (1-10 μM=0.41-4.1 μg/ml) of CAMPinducer, forskolin (FK) and c-Src kinase inhibitor geldanamycin (GM) 24hours prior to cell proliferation assessment. Tamoxifen (TX) was used inparallel as positive control. Cell proliferation was measured using³H-thymidine incorporation. The study was done in triplicate and acontrol was used with solvent only. Control received solvent only at<0.1% ethanol. Results are shown in Table 2 and FIG. 1. TABLE 2Dose-Response Effect on E₂ Induced MCF-7 Cell Proliferation ³H-Thymidineincorporation/μg protein Treatment (% of control) % protection Control100 E₂ (10 nM) 290 proliferation In the presence of 10 nM E₂ FK 1 μM 3466 FK 5 μM 22 78 FK 10 μM 14 86 TX 1 μM 79 21 TX 5 μM 54 46 TX 10 μM 2575 GM 1 ng/ml no effect none GM 5 ng/ml no effect none GM 10 ng/ml 94 6

[0049] Table 2 illustrates that a) cAMP/PKA cascade, and estrogenreceptor pathway are involved in the cancer cell proliferation, b) theantiestrogenic action of TX and FK is dose-dependent, and c) GM, thec-Src kinase inhibitor, at 10 ng/ml did not inhibit the E₂-induced cellproliferation.

Example 3

[0050] Effect of FK or GM on TX Efficacy Against E₂-Induced CellProliferation

[0051] MCF-7 cells were cultured in growth DMEM medium supplemented with10% fetal 10 bovine serum (FBS). At 85% confluence, cells weresub-cultured in 5% FBS serum stripped medium, phenol red free for 24hours prior to the treatment of the test chemical. Cells were treatedwith 10 nM E₂ (=2.7 ng E₂/ml) with and without two concentrations of FK(1 and 5 μM =0.41 and 2.05 pg FK/ml). In another experiment cells weretreated with 10 nM E₂ (=2.7 ng E₂/ml) with three concentraion of GM (1,5 and 10 ng/ml) 24 hours prior to cell proliferation assessment.Tamoxifen (TX) was used in parallel as positive control. Cellproliferation was measured using ³H-thymidine incorporation. The studywas done in triplicate and a control was used with solvent only. Controlreceived solvent only at <0.1% ethanol. Results are shown in Tables 3and 4 and FIGS. 2 and 3.

[0052] The data in Table 3 shows that the synergistic action between TXand FK depends on the ratio between the two chemicals when they areapplied as a mixture. The ratio recommended is 1 part TX: 3.7 parts FK,based on μg of active ingredient per ml. In addition, although GM alonedid not antagonize the estrogenic activity of E2, it was able toincrease the efficacy of TX significantly (Table 4). Our data showedthat when 10 ng GM/ml was mixed with 5 μM TX (=2.8 ug TX/ml), a 75%protection (vs 46% protection by 5 μM TX alone) was revealed (Table 4).Another confirmation for the synergistic action of GM on TX againstE₂-induced breast cancer cell growth emerged from the data in FIG. 2.These data illustrate that 1 μM of TX provides an approximate 25%protection against the biological activity of E₂. In the presence of 10ng GM/ml, 45% increase in the antiestrogenicity of 1 μM TX (=0.56 ugTX/ml) was found. Interestingly, 5 μM TX alone induced 45% protectionagainst the E₂-induced cancer cell growth. Thus, the concentration of TXcan be reduced by 20 percent to minimize (if not abolish) detrimentalside effects without adversely affecting efficacy, as shown in FIG. 3which illustrates the synergistic relationship between TX and FK or GM.TABLE 3 Dose-Response Effect on E₂ Induced MCF-7 Cell Proliferation³H-Thymidine incorporation/μg protein Treatment % of control %protection Control 100 E₂ (10 nM) 290 proliferation In the presence of10 nM E₂ FK 1 μM 34 66 FK 5 μM 22 78 FK 10 μM 14 86 TX 1 μM 79 21 TX 5μM 54 46 TX 10 μM 25 75 GM 1 ng/ml no effect none GM 5 ng/ml no effectnone GM 10 ng/ml 94 6 With 1 μM FK: FK + TX 1 μM + E₂ 43 57 FK + TX 5μM + E₂ 21 79 FK + TX 10 μM + E₂ 13 87 With 5 μM FK: FK + TX 1 μM + E₂21 79 FK + TX 5 μM + E₂ 12 88 FK + TX 10 μM + E₂ 9 91

[0053] TABLE 4 Interaction Between TX and GM on MCF-7 Cell Proliferation³H-Thymidine incorporation/μg protein Treatment (% of control) %protection E₂ 10 Nm 100 proliferation GM 1 ng/ml 100 0 GM 5 ng/ml 100 0GM 10 ng/ml 90 10 With 5 μM TX: + E₂ 54 46 + E₂ + GM 1 ng/ml 54 46 +E₂ + GM 5 ng/ml 50 50 + E₂ + GM 10 ng/ml 25 75 E₂ + FK 5 μM + GM 10ng/ml + TX 31 69 5 μM

Example 4

[0054] Effect of cAMP Inducer on Efficacy and Stability of TamoxifenMCF-7 cells were cultured in growth DMEM medium supplemented with 10%fetal bovine serum (FBS). At 85% confluence, cells were sub-cultured in5% FBS serum stripped medium, phenol red free for 24 hours prior to thetreatment of the test chemical. Cells were treated with 10 nM E₂ (=2.7ng E₂/ml) with and without a mixture of 1 μM FK and 1 μM TX. Cell growthwas measured 1, 2 and 5 days following treatment. Cell growth wasdetermined by cell count using trypan blue exclusion. The study was donein duplicate and a control was used with solvent only. Control receivedsolvent only at <. 1% ethanol. Results are shown in FIGS. 4 and 5.

[0055]FIG. 4 illustrates that when the efficacy of TX and FK against E2action was calculated based on the control value the following wasfound: (a) TX (1 μM =0.56 ug TX/ml) alone was able to antagonize the E₂action up to 48 hr after treatment and (b) this action of TX wassustained up to 4-5 days in the presence of 1 μM FK (FIG. 4).

[0056]FIG. 5 shows that when the efficacy of TX and FK was calculated aspercentage of the E₂-induced cell growth value the same conclusion wasfound except more stability and protection by TX was found in thepresence of FK.

Example 5

[0057] Effect of Forskolin on Tamoxifen efficacy

[0058] MCF-7 cells were cultured in growth DMEM medium supplemented with10% fetal bovine serum (FBS). At 85% confluence, cells were sub-culturedin 5% FBS serum stripped medium, phenol red free for 24 hours prior tothe treatment of the test chemical. Cells were treated with and withouta mixture of either 5 μM FK (2.05 μg FK/ml) and 1 μM TX (=0.56 ug TX/ml)or a mixture of 1 μM TX (=0.56 ug TX/ml) and 1 μM FK. Cell growth wasmeasured 5 days following treatment. Cell growth was determined by cellcount using trypan blue exclusion. The study was done in duplicate and acontrol was used with solvent only. Control received solvent only at<0.1% ethanol. Results are shown in FIGS. 6 and 7.

Example 6

[0059] Effect of c-Src Kinase Inhibitor GM on Tamoxifen Efficacy

[0060] MCF-7 cells were cultured in growth DMEM medium supplemented with10% fetal bovine serum (FBS). At 85% confluence, cells were sub-culturedin 5% FBS serum stripped medium, phenol red free for 24 hours prior tothe treatment of the test chemical. Cells were treated with and withouta mixture of either 5 ng GM/ml and 5 μM TX (=2.8 ug TX/ml) or a mixtureof 10 ng GM/ml and 5 μM TX (=2.8 ug TX/ml). Cell growth was measured 5days following treatment. Cell growth was determined by cell count usingtrypan blue exclusion. The study was done in duplicate and a control wasused with solvent only. Control received solvent only at <0.1% ethanol.Results are shown in FIG. 8.

Example 7

[0061] Effect of mitogen activated protein kinases inhibitor PD98059 onTX efficacy

[0062] MCF-7 cells were cultured in growth DMEM medium supplemented with10% fetal bovine serum (FBS). At 85% confluence, cells were sub-culturedin 5% FBS serum stripped medium, phenol red free for 24 hours prior tothe treatment of the test chemical. Cells were treated with and withouta mixture of 5 μM TX (=2.8 ug TX/ml) and 2 μM PD98059. Cell growth wasmeasured 5 days following treatment. Cell growth was determined by cellcount using trypan blue exclusion. The study was done in duplicate and acontrol was used with solvent only. Control received solvent only at<0.1% ethanol. Results are shown in FIG. 9. p The above Examples show,inter alia, that (a) signal transduction cascades are vital in thecancer cell growth and cell proliferation and (b) CAMP inducers (e.g.,FK), c-Src kinase inhibitors (e.g., GM) and MAPK inhibitors (e.g.,PD98059) synergize the anticancer activity of tamoxifen in breast cancercells. In general, one or more signal transduction modulators may beused in combination with plant essential oil compounds and derivativesthereof to provide anti-proliferative, anti-estrogenic and/oranti-mitogenic compositions for prophylactically and/or therapeuticallytreating soft tissue cancers.

[0063] Although illustrative embodiments of the present invention havebeen described in detail, it is to be understood that the presentinvention is not limited to those precise embodiments, and that variouschanges and modifications can be effected therein by one skilled in theart without departing from the scope and spirit of the invention asdefined by the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising at leastone plant essential oil compound and at least one signal transductionmodulator.
 2. The pharmaceutical composition of claim 1, wherein theplant essential oil compound is selected from the group consisting ofaldehyde C16 (pure), amyl cinnamic aldehyde, amyl salicylate, anisicaldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde, cinnamicalcohol, α-terpineol, carvacrol, carveol, citral, citronellal,citronellol, p-cymene, diethyl phthalate, dimethyl salicylate,dipropylene glycol, eucalyptol (cineole), eugenol, iso-eugenol,galaxolide, geraniol, guaiacol, ionone, d-limonene, menthol, methylanthranilate, methyl ionone, methyl salicylate, a-phellandrene,pennyroyal oil perillaldehyde, 1- or 2-phenyl ethyl alcohol, 1- or2-phenyl ethyl propionate, piperonal, piperonyl acetate, piperonylalcohol, D-pulegone, terpinen4-ol, terpinyl acetate, 4-tertbutylcyclohexyl acetate, thyme oil, thymol, metabolites oftrans-anethole, vanillin, and ethyl vanillin.
 3. The pharmaceuticalcomposition of claim 1, wherein the signal transduction modulator isselected from the group consisting of cyclic adenosine monophosphate(CAMP)/cAMP-dependent protein kinase, tyrosine kinase, calciumphospholipid-dependent protein kinase (PKC), mitogen activated proteinkinase family members, calcium-calmodulin-dependent protein kinase, andgrowth factor receptor inhibitors.
 4. The pharmaceutical composition ofclaim 1, further comprising tamoxifen.
 5. The pharmaceutical compositionof claim 1, further comprising a pharmaceutically acceptable carrier. 6.The pharmaceutical composition of claim 1, wherein the soft tissuecancer is breast cancer.
 7. The pharmaceutical composition of claim 2,wherein the plant essential oil compound is eugenol.
 8. Thepharmaceutical composition of claim 2, wherein the plant essential oilcompound is thymol.
 9. The pharmaceutical composition of claim 2,wherein the plant essential oil compound is isoeugenol.
 10. Thepharmaceutical composition of claim 2, wherein the plant essential oilcompound is benzyl alcohol.
 11. The pharmaceutical composition of claim2, wherein the plant essential oil compound is carvacrol.
 12. Thepharmaceutical composition of claim 2, wherein the plant essential oilcompound is cinnamic alcohol.
 13. The pharmaceutical composition ofclaim 2, wherein the plant essential oil compound is cinnamic aldehyde.14. The pharmaceutical composition of claim 2, wherein the plantessential oil compound is citronellal.
 15. The pharmaceuticalcomposition of claim 2, wherein the plant essential oil compound istrans-anethole.
 16. A method for treating cancer, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the composition of claim 1.